Combinatorial approach to organelle-targeted fluorescent library based on the styryl scaffold

Article abstract of DOI:10.1021/ja027587x

The first fluorescent styryl dye library with a broad color range was synthesized by combinatorial condensation of various aldehydes and methyl pyridinium compounds, and their applications as organelle specific staining probes were demonstrated. Copyright

Full text of DOI:10.1021/ja027587x

Published on Web 01/14/2003
Combinatorial Approach to Organelle-Targeted Fluorescent Library Based on
the Styryl Scaffold
Gustavo R. Rosania,*,† Jae Wook Lee, Liang Ding, Hai-Shin Yoon, and Young-Tae Chang*
Department of Chemistry, New York UniVersity, New York, New York 10003, and Department of Pharmaceutics,
College of Pharmacy, UniVersity of Michigan, Ann Arbor, Michigan 48109
Received July 8, 2002 ; E-mail: yt.chang@nyu.edu
Scheme 1. Synthesis of styryl dyes
Fluorescent compounds have attracted attention due to their broad
application1 coupled with highly sensitive and specific detection
methods.2 Styryl dyes are a class of fluorescent, lipophilic cations
that have been used as mitochondrial labeling agents and membrane
voltage-sensitive probes of cellular structure and function.3 Because
of the electrochemical potential across the mitochondrial inner
membrane, these lipophilic cations can accumulate in mitochondria
according to the Nernst equation.4 Nevertheless, the affinity of some
styryl molecules for cellular macromolecules such as DNA prompts
the question as to what structural or physicochemical features of
these molecules can actually confer specific mitochondrial localiza-
tion, and whether certain derivatives could be developed as probes
for other organelles or macromolecules.5
While rational design of compounds with specific emission
wavelengths and high quantum yields is difficult, the combinatorial
approach has been reported to be quite powerful in developing
fluorescent libraries.6 For microscopic imaging and flow cytometry
applications, it is often desirable to obtain fluorescent compounds
that excite or emit in a specific color range. Nevertheless, the
spectral properties and potential applications of the reported
combinatorial fluorescent libraries are still limited. In this com-
munication, we report the first combinatorial wide-color range
fluorescent toolbox and its potential application as organelle-specific
probes.
Our fluorescent library is based on the styryl scaffold, synthesized
by the condensation of 41 aldehydes (A) and 14 pyridinium (2- or
4-methyl) salts (B) (Scheme 1). For building blocks, we chose
commercially available aldehydes (A) containing functionalities of
various sizes, conjugation lengths, and electron-donating or -with-
drawing capabilities. N-methyl pyridinium iodide compounds (B)
were synthesized by the methylation of commercially available 2-
or 4-methylpyridine derivatives using methyl iodide.7 The conden-
sation of A and B with a secondary amine catalyst8 was performed
in 96-well plates, and the dehydration reaction was accelerated by
microwave irradiation for 5 min to give 10-90% conversion. The
resulting library compounds were analyzed by LC-MS equipped
with diode array and fluorescence detectors, and a fluorescence
plate-reader to determine the absorption and emission maximum
(λex and λem), and the emission colors are summarized in Table 1.
It can be easily visualized that this styryl dye library covers a
broad color range from blue to long red, representing practically
all the visible colors! This should be attributed to the structural
diversity as discussed above. It is noteworthy that further purifica-
tion is not required for primary analysis, as the fluorescent properties
of the products are easily distinguishable from those of left-over
building blocks A and B (weak fluorescence or much shorter λex
and λem). Furthermore, the synthesis was designed so that the
reaction mixture can be used directly in biological screening; toxic
catalysts (such as strong acid, base, metals) were avoided, and most
of the low-boiling point solvent and catalyst (pyrrolidine) were
removed during the microwave reaction, leaving only DMSO, the
common solvent for biological sample preparation.
Without further purification, the library compounds were incu-
bated with live UACC-62 human melanoma cells growing on glass
bottom 96-well plates, and the localizations of the different
compounds in the cells were determined using an Axiovert (Zeiss)
microscope (λex ) 405, 490, and 570 nm; λem > 510 nm) with a
100× Zeiss oil immersion objective. It was found that 119 out of
276 fluorescent compounds localize to specific subcellular compart-
ments (i.e., mitochondria, ER (endoplasmic reticulum), vesicles,
nucleoli, chromatin, cytoplasm, or granules). The images in Figure
1 show cells stained with selected fluorescent compounds. Previous
studies have established that there is large voltage between the inside
of the mitochondria and the cytosol, and compounds with strong
polarizability and charged compounds can interact strongly with
the mitochondrial membrane. Considering that our library com-
† University of Michigan, College of Pharmacy.
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J. AM. CHEM. SOC. 2003, 125, 1130-1131
10.1021/ja027587x CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Emission Colors of the Fluorescent Compounds from the
Styryl Dye Library [(a) Components in Building Block A, (b)
Components in Building Block B]a
Table 2. Localization Distribution of the Organelle Specific Styryl
Dyes [(#) Nuclear, (*) Nucleolar, (() Mitochondria, (b) Cytosolic,
(×) Endoplasmic Reticular (ER), (9) Vesicular, (2) Granular]a
a Row a is aldehyde only.
a Row a is aldehyde only.
binding in a larger view, some compounds may be DNA, RNA, or
protein-specific binding probes. Further studies will be carried out
on the compounds that bind specifically to these macromolecules
and those compounds that are sensitive to the cellular environment,
to elucidate structural features of the styryl molecules responsible
for their organelle selectivity and optical properties.
Supporting Information Available: All experimental procedures
and chemical and biological data (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
References
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Figure 1. Images of representative localizations (bar ) 10 µm): nucleolar
(I19); nuclear (H28); mitochondria (A12); cytosolic (I37); vesicular (H12);
granular (B41); reticular (J37); multilabeled: nucleolar (I19, red), granular
(34, blue), mitochondrial (B24, green).
pounds are positively charged, it is not surprising that 64 out of
119 selected compounds localize specifically to mitochondria.
Again, owing to the diversity of the molecular structure, some
compounds targeted organelles other than mitochondria. This
encrypted interesting structure-localization relationships (SLR),
which can lead to rational design of molecular probes for cellular
components and opened the chance of multicolor labeling using
our toolbox (Table 2).
Combinatorial chemistry has grown to be one of the most
powerful tools in new drugs and materials discovery, and our
combinatorial approach for organelle-targeted fluorescent dyes
further demonstrates its prowess. In addition to organelle-specific
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